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Poult Sci 2008. 87:1248-1252. doi:10.3382/ps.2006-00440
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ENVIRONMENT, WELL-BEING, AND BEHAVIOR

The Stress and Fear Levels of Microwave Toe-Treated Broiler Chickens Grown with Two Photoperiod Programs

B. Wang*, B. M. Rathgeber{dagger},1, T. Astatkie{ddagger} and J. L. MacIsaac§

* Plant & Animal Sciences, Nova Scotia Agricultural College, Truro, Nova Scotia B2N 5E3, Canada; {dagger} Crops and Livestock Research Centre, Charlottetown, Prince Edward Island, Canada C1A 7M8, Canada; {ddagger} Department of Engineering, Nova Scotia Agricultural College, Truro, Nova Scotia, B2N 5E3, Canada; and § Atlantic Poultry Research Institute, Truro, Nova Scotia, B2N 5E3, Canada

1 Corresponding author: rathgeberb{at}agr.gc.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The current study was conducted to investigate the influence of microwave toe treatment and an increasing photoperiod on stress and fear levels in broiler chickens. Upon delivery from the hatchery, the toe tips of 364 male and 364 female broilers were exposed to microwave energy to restrict claw growth, whereas the same numbers of birds retained intact toes. Birds from each sex and toe treatment were grown under 23 h of light or increasing photoperiods, with 4 replicates of each treatment combination. Elevated activity of plasma creatine kinase (CK) and increased heterophil to lymphocyte (H/L) ratios were employed as indicators of stress. Tonic immobility (TI) was conducted on d 10, 22, and 36 and used as an index of fear response. Males had lower plasma CK activity and H/L ratios (P < 0.05). Birds on 23 h of light had a higher activity of plasma CK (P < 0.05) but similar H/L ratios compared with birds on the increasing photoperiod. Microwave toe treatment did not affect the activity of plasma CK or H/L ratios. Photoperiod, microwave toe treatment, or sex did not affect the number of attempts required to induce TI. The increasing lighting program decreased the duration of TI on d 10 (P < 0.05), had no effect on d 22, and increased duration of TI on d 36 (P < 0.05). There was no microwave toe treatment or sex effect on the duration of TI at any ages tested.

Key Words: microwave toe treatment • photoperiod • stress • fear • broiler


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Stress and fear have received much attention in the poultry industry in recent years due to growing concerns for bird welfare. Stress and fear may have deleterious effects on growth, reproduction, and meat quality of birds (McKee and Sams, 1997; Tankson et al., 2001; Mashaly et al., 2004). The stress response begins with the central nervous system; 4 general biological defense responses follow, including the behavioral response, the autonomic nervous system response, the neuroendocrine response, and the immune response (Moberg, 2000).

In the broiler chicken industry, birds have traditionally been kept on a continuous or nearly continuous photoperiod schedule (23 or 24 h of light/d) throughout the growing period to maximize feed intake and growth rate (Morris, 1967; Beane et al., 1979). However, considerable evidence suggests that there are benefits to bird welfare when broilers are kept on short or moderate day lengths, including lower physiological stress and lower levels of fear (Gordon, 1994; Davis et al., 1999; Rozenboim et al., 1999). Increasing photoperiod programs, which provide short photoperiod early in life and increased day length as a broiler ages, have been associated with a reduction in the incidence of heart disease and leg problems (Classen and Riddell, 1989; Classen et al., 1991; Rozenboim et al., 1999). However, broilers on increasing photoperiods may have a higher number of carcass scratches inflicted by toenails of other birds due to increased bird activity (Blair et al., 1993).

Currently, the turkey industry treats the toes of day-old poults with microwave energy to restrict claw growth and reduce carcass scratches. Before the development of microwave toe technology, researchers experimented with hot blade beak trimming equipment to remove the tip of the toe. Compton et al. (1981) used a hot blade to trim the toes of White Leghorn chicks and proposed that toe trimming posed an initial stress to the birds as indicated by increased plasma corticosterone (CS). The pain caused by toe treatment early in life may easily be outweighed by the chronic pain of lacerations and resulting infections to the skin of birds later in the rearing period. Honaker and Ruszler (2004) reported that microwave toe-treated Leghorn layers displayed lower levels of fear as measured by the tendency to panic in the presence of a human. Satterlee et al. (1985) also found toe-treated hens to be less active and fearful. To the authors’ knowledge, there have been no publications on the effect of microwave toe treatment on stress or fear levels in broiler chickens.

There are several measurements that have been used to examine stress and fear levels. The heterophil to lymphocyte (H/L) ratio has been established as a widely accepted indicator for determining chronic stress in poultry (Gross and Siegel, 1983). In chickens exposed to environmental stressors, the number of lymphocytes decreases and the number of heterophils increases, thus increasing H/L ratios (Jones et al., 1988). In addition, the hypothalamic-pituitary-adrenocortical axis is activated and increases plasma CS concentrations. It has been found that long-term elevation of plasma CS concentrations may impair leucocytic responsiveness (Jones et al., 1988), whereas the activation of the hypothalamic-pituitary-adrenocortical axis inhibits humoral and cell-mediated immunity (Davison et al., 1983; Siegel et al., 1983). In recent years, the elevated activity of plasma creatine kinase (CK) has been observed in stressed birds (Mitchell and Sandercock, 1995). In broiler chickens, CK is released into circulation following changes in the permeability of the sarcolemma in response to various pathologies and exposure to environmental stressors (Mitchell and Sandercock, 1995). Tonic immobility (TI) reaction has also been considered as a reliable index of fearfulness in birds (Gallup, 1979). High susceptibility to TI and a long duration of TI are signs of a high level of fear (Jones, 1986, 1987). The objective of this study was to investigate the influence of microwave toe treatment and an increasing photoperiod on stress and fear levels of broiler chickens, as indicated by plasma CK activity, H/L ratio, and TI.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Experimental Conditions

Two replicate trials (fall and winter) were conducted with 728 male and 728 female broilers each. Sexed, day-old broilers (Ross 308) were obtained from a commercial hatchery and were placed in separate pens in a confinement house with untreated wood shavings as litter. Animal care was provided according to the standards of the Canadian Council on Animal Care (1993). For each trial, male and female broilers were held in separate rooms and each room was separated by a black plastic divider into 2 sections for 2 photoperiods. In each room, birds were randomly assigned to 1 of 16 pens, with 49 birds per pen in room 1 and 42 birds per pen in room 2 to maintain a stocking density of 0.07 m2·bird–1.

Feed and water were available ad libitum with a nipple drinker system in each room and one suspended tube feeder per pen. Box feeders were used to provide additional feed in the first week. All the birds were given the same starter, grower, and finisher rations. The starter diet contained 23% crude protein and 3,050 kcal of ME·kg–1 and was fed as mash from d 0 to 13. The grower diet contained 20% crude protein and 3,150 kcal of ME·kg–1 and was fed as mash from d 14 to 23. The finisher diet contained 18% crude protein and 3,200 kcal of ME·kg–1 and was fed as pellets from d 24 to 38. The brooding temperature was 30 to 32°C from d 0 to 7 then reduced 3°C per week until it reached 21°C where it remained for the rest of the trial.

Half of the birds from each sex were toe treated using a Microwave Claw Processor (equipment developed by Nova-Tech Engineering Inc., Willimar, MN) upon delivery from the hatchery. Chicks were turned upside down, and their legs were inserted into shackles of the machine with the ventral side of the bird toward the operator. Each of the 3 front toes was pulled into place by a vacuum and treated with microwave energy for 0.8 s. Light was provided by incandescent bulbs mounted at approximately 2.0 m above the floor level. All birds were subjected to the same light intensity. Light intensity was measured at the bird head level with a light meter (Cal-Light 400, the Cooke Corporation, Romulus, MI) and adjusted by rheostat. All broilers were given 24 h of light for the first 3 d, after which a 23-h light program and an increasing lighting (IL) program were applied (Table 1Go).


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  		Table 1. Experimental lighting programs used to rear toe-treated and intact-toe male and female broilers for 38 d
 
Blood Sampling

At 21 and 35 d of age, blood samples were collected from 3 randomly selected birds per pen; birds sampled on d 21 were banded and sampled again on d 35. Approximately 3.5 mL of blood was collected via venipuncture of the brachial vein into a 7.0-mL Vacutainer tube containing sodium heparin as anticoagulant. Blood samples were immediately placed on ice until they were ready for centrifugation. Two drops of blood were collected from 2 of the 3 birds using heparinized microcapillary tubes and were smeared onto duplicate glass slides for determining H/L ratio. Blood samples were centrifuged at 1,600 x g at 4°C for 15 min and plasma samples transferred into microcentrifuge tubes and frozen at –80°C until analyzed.

Tonic Immobility Test

On d 10, 22, and 36, two birds from each pen were randomly selected for TI. Birds used for blood sampling on d 21 were not used for TI. Tonic immobility was induced by inverting the bird on its back in a plastic box containing 10 cm of untreated wood shavings. To induce immobility the handler rested one hand lightly on the bird’s breast and the other over its head. The bird was restrained for 15 s. If the bird remained immobile for 10 s, TI was considered to be induced. If the bird righted itself before this minimum time expired, the procedure was repeated until the immobility response was induced. A maximum of 10 inductions were performed. If the bird failed to be induced, another bird was used. If the bird did not show a righting response over the 10-min period, the bird was returned to the pen and a maximum duration of 600 s was given for righting time. This could potentially bias the results, but it was decided that a maximum duration may be required to conduct the procedure in a reasonable time period. The number of times needed to induce immobility and the duration of immobility were recorded. Birds on which this procedure was performed were not identified to avoid selection bias at a later age; however, the likelihood of this occurring in a pen of 40-plus birds would be quite small.

Laboratory Analysis

The activity of plasma CK was measured by the Veterinary Diagnostic Laboratory of the Nova Scotia Department of Agriculture and Fisheries using a commercial assay kit (Bayers Inc., Toronto, Ontario, Canada). Blood smears were fixed and stained using Hema 3 stain set (Fisher Diagnostics, Fisher Scientific Company LLC, Middletown, VA) and observed under a light microscope. One hundred leukocytes, including granular (heterophils, eosinophils, basophils) and nongranular (lymphocytes, monocytes) cells, were randomly selected and counted. Two slides were counted per bird, and the mean H/L ratio was calculated.

Statistical Analysis

The experiment was conducted as a split-plot factorial design, with 4 blocks (2 trials and 2 rooms per trial), 2 whole-plot treatments (23 h and increasing photoperiod), and 4 subplot treatments (factorial of treated and intact toes, and male and female birds). The response variables included plasma CK activity, H/L ratio, and time-to-induction duration of TI. Data were analyzed as repeated measures using PROC MIXED procedure in SAS software (SAS Institute, 1999), with a level of significance of {alpha} = 5%. Data transformation was conducted when the distribution of the error terms was not normal. When a significant difference was found among main effects or their interactions, least squares means comparison tests in SAS were used to compare the means.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Plasma Creatine Kinase Activity

Microwave toe treatment did not influence plasma CK activity, although the enzyme was affected by photoperiod, sex, and age (Table 2Go). Birds on 23 h of light had a higher activity of plasma CK than birds on IL at 21 and 35 d. There was a significant difference in plasma CK activities between sexes; males showed lower plasma CK activity at 21 and 35 d. All birds had a significantly higher activity of plasma CK at 35 d than at 21 d. Marked elevations in plasma CK activity have been reported in several pathological muscle conditions in poultry including growth-associated and stress-induced myopathies (Mitchell et al., 1992; Mitchell and Sandercock, 1994, 1995) and have been associated with heat stress (Mitchell and Sandercock, 1995) and water-immersion stress (Dzaja et al., 1996). Our findings that broilers at 35 d of age had a significantly higher activity of plasma CK than at 21 d of age are consistent with Hocking et al. (1998) who proposed that increases in plasma CK activity in older birds may reflect age- or size-dependent increases in muscle cell metabolism and turnover. The increased activity of plasma CK for birds on 23 h of light in our study suggests that this lighting program may have detrimental effects on the meat quality of broilers because elevated CK activity has been well established as a reliable indicator of skeletal muscle damage in birds (Hamburg et al., 1992).


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  		Table 2. Effect of lighting, microwave toe treatment, and sex on plasma creatine kinase (CK) activity of broilers grown to 35 d of age
 
Heterophil to Lymphocyte Ratio

Neither microwave toe treatment nor lighting program had a significant effect on the H/L ratio at d 21 or 35 (Table 3Go). However, this does not exclude the possibility that stress occurs at an early age due to microwave toe treatment. Compton et al. (1981) examined blood CS levels and found that toe-trimming posed an initial stress to cage reared Leghorns at 7 d of age. Puvadolpirod and Thaxton (2000) found that the first response to stress was an elevated plasma CS level at 2 h, lasting for 6 d, and an elevated H/L ratio at d 2, lasting for 10 d. In our study, it was not possible to collect adequate volumes of blood at 7 d of age. If the H/L ratios had increased early in life due to an initial stress from toe treatment, the increased H/L ratios were not maintained by 21 d of age when the first blood samples were taken. There was a significant difference in the H/L ratio between sexes, with females having a greater H/L ratio than the males.


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  		Table 3. Effect of lighting, microwave toe treatment, and sex on heterophil/lymphocyte (H/L) ratio of broilers at 21 and 35 d of age
 
That 23 h of light did not increase H/L ratios in our study supports the findings of Campo and Davila (2002), who measured H/L ratios in 36-wk hens exposed to lighting programs of 23L:1D, 14L:10D, or 18.5L:5.5D and proposed that there was no evidence of an increased stress response. Blair et al. (1993) also reported that the H/L ratio in broilers was not affected by photoperiod. It has been postulated that continuous lighting reduces the opportunity for rest and sleep, thereby increasing physiological stress (Gordon, 1994). Zulkifli et al. (1998) found that broiler chickens illuminated 24 h per d had greater H/L ratios, whereas Vo et al. (1998) noted an increase in H/L ratio for broilers housed under continuous photoperiod.

The Tonic Immobility Test

Photoperiod had a significant effect on TI (Table 4Go). Birds on 23 h of light had a longer duration of TI on d 10, although the trend reversed on d 36, when the birds on IL were found to have a longer duration of TI. This may indicate that birds on IL could not easily adjust to change of day length in later life, resulting in a higher level of fear of humans. In growing broilers, continuous light has been found to increase level of fear of humans as indicated by a longer duration of TI (Sanotra et al. 2002). Age had a significant effect on TI, with the induction of TI on d 10 being significantly shorter than that on d 22 and 36 (P < 0.05). This is consistent with findings of Heiblum et al. (1998), who found TI was poorly developed during the first 3 d of life.


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  		Table 4. Effect of lighting, microwave toe treatment, and sex on the duration of tonic immobility (TI) of broilers at 21 and 35 d of age
 
Tonic immobility was not affected by microwave toe treatment or sex. To the authors’ knowledge, there have been no reports on the effect of toe treatment on TI response. Honaker and Ruszler (2004) evaluated the level of fear of microwave toe-treated Leghorn hens and found lower objective fearfulness scores on toe-treated birds. Compton et al. (1981) also reported that toe-treated cage-reared Leghorn hens were less fearful. Owings et al. (1972) noted less fighting and flying of toe-treated turkeys. It appears that contact with humans early in life due to the toe-treatment procedure could decrease the level of fear to humans in later life. The relationship between sex and TI response in birds is unclear. Differences between sexes have been observed in the responses of domestic chicks and adolescent birds to a variety of potentially frightening situations, and this dichotomy has been attributed to greater fearfulness in males (Banks et al., 1979; Jones, 1985). However, no sex differences in the duration of TI were found in Japanese quail and domestic chicks (Gallup, 1974; Benoff and Siegel, 1976).

Results from this study demonstrate that microwave toe treatment does not influence overall levels of stress and fear of humans for broilers from 10 to 35 d of age. The effects of photoperiod are less clear; an increasing photoperiod program may reduce the level of stress and fear of humans early in life but increase the level of fear later in life.


   ACKNOWLEDGMENTS
 
Special thanks are extended to R. Mekers, R. Currie, and K. Budgell (Nova Scotia Agricultural College, Truro, Nova Scotia, Canada) for their assistance in the production facilities. The authors would like to thank Nova-Tech Engineering for supplying the microwave toe treatment equipment, and the Poultry Industry Council and Technology Development for funding toward this project.

Received for publication December 16, 2006. Accepted for publication March 20, 2008.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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